The intermediate, designated X, formed during the self-assembly reaction of the tryosyl radical/m-oxo-bridged diferric cofactor in the R2 subunit of E. coli ribonucleotide reductase (RNR), is directly involved in the oxidation of Y122 to the catalytically essential Y122. S-band EPR studies monitored the formation of X in both WT and Y122F R2 help reveal that X contains two oxygen atoms. Both are initially derived from molecular oxygen with one present as a mu-oxo bridge and one as the terminal aqua ligand; with time the latter of these atoms exchanges with the solvent. Water and molecular oxygen isotopically enriched with O17 were used in this study.